TRAF3 is a novel tumor suppressor gene identified in a variety of human B lymphoma, including splenic marginal zone lymphoma, B cell chronic lymphocytic leukemia and mantle cell lymphoma, as well as multiple myeloma (MM). To explore the role of TRAF3, we recently generated a new genetically modified mouse model that has the TRAF3 gene specifically deleted in B cells (B-TRAF3-/- mice). We found that TRAF3 deletion results in prolonged survival of mature B cells, which eventually leads to spontaneous development of B lymphomas in mice. This proposal aims to understand how TRAF3 inactivation promotes oncogenic B cell survival. We first found that the mechanism of aberrant B cell survival mediated by TRAF3 inactivation is fundamentally different from that of normal B cell survival induced by physiological stimuli. To identify novel targets of TRAF3 inactivation, we performed microarray analysis to compare the global gene expression profiles of splenic B cells purified from young, tumor-free B-TRAF3-/- mice and littermate control mice. Strikingly, a number of genes identified in our microarray analysis are implicated in subcellular organelle or vesicle trafficking, including Rhbdf1, Rasgrp3, Ehd1, and Kif11, etc. Current evidence indicates that many survival proteins are trafficking between two important organelles, endoplasmic reticulum (ER) and mitochondria, and that the balance between mitochondrial levels of pro-survival proteins and anti-survival proteins controls the threshold of cell death. Interestingly, or preliminary results demonstrated that mitochondrial levels of the anti-survival protein Bak were decreased, and mitochondrial levels of the pro- survival protein Mcl-1 were increased in premalignant TRAF3-/- B cells. However, total cellular levels of Bak and Mcl-1 were not altered in these cells. Based on our new findings, we will test the central hypothesis that TRAF3 inactivation modulates the trafficking of Bak and Mcl-1 between ER and mitochondria to increase the threshold of cell death in B cells. To address this, we propose complementary studies using B-TRAF3-/- mice and human patient-derived multiple myeloma cell lines with TRAF3 deletions or mutations as model systems. In Aim 1, using microarray analysis and a stringent prioritization scheme, our preliminary results led us to focus on a novel target of TRAF3, Rhbdf1, which is implicated in ER protein trafficking. To delineate the causal role of Rhbdf1 in oncogenic B cell survival, we will employ lentiviral shRNA vector-mediated knockdown, ectopic overexpression, and mutagenesis of Rhbdf1. In Aim 2, we will perform extensive biochemical fractionation, confocal microscopic imaging, and co-immunoprecipitation studies to understand how TRAF3 inactivation and Rhbdf1 modulate the trafficking of Bak and Mcl-1 between ER and mitochondria. Furthermore, we will interrogate how TRAF3 signaling interplays and cross-talks with ER stress responses. In summary, this study will provide new insights into the exact mechanisms of TRAF3 inactivation-mediated oncogenic B cell survival, and will open up new avenues for the treatment of B lymphoma and MM. PUBLIC HEALTH RELEVANCE: Lymphoma is the 5th most common human cancer with approximately 21,000 annual deaths in the United States. TRAF3 is a novel tumor suppressor gene identified in a variety of human B lymphoma, including splenic marginal zone lymphoma, B cell chronic lymphocytic leukemia and mantle cell lymphoma, as well as multiple myeloma. This proposal aims to investigate a novel target of TRAF3, Rhbdf1, and a new signaling mechanism that connect TRAF3 inactivation to oncogenic B cell survival. To address the gaps in knowledge, we propose complementary studies using a new mouse B lymphoma model with the TRAF3 gene specifically deleted in B lymphocytes and human patient-derived multiple myeloma cell lines with TRAF3 deletions or mutations. Information gathered from the proposed studies will advance the understanding of the pathological mechanisms underlying B cell oncogenesis, and will open up new avenues for the development of treatment strategies to combat B lymphoma and multiple myeloma.